Herpes simplex virus (HSV) is a significant human pathogen infecting 65%-90% of the population worldwide and primarily causing oral (HSV-1) or genital (HSV-2) mucocutaneous ulcers. More serious infections can result in life-threatening encephalitis, and keratitis from HSV infection is the leading cause of blindness in the United States. HSV-2 is one of the most prevalent sexually transmitted diseases worldwide; it is also a significant co-factor for HIV spread. Several antivirals are approved for the treatment of HSV infections, but their efficacy is limited to the period before latency is established and can be compromised by the emergence of drug resistance. It is, therefore, important to investigate new drug targets and treatment strategies. The cleavage and packaging of the viral DNA into preassembled capsids is a critical step in the life cycle of herpes virus replication; understandin this process has an important practical implication because it provides a rich target for developing novel anti-herpes virus strategies. This DNA cleavage and packaging reaction is mediated by terminase, a virally-encoded enzyme composed of pUL15, pUL28 and pUL33 in HSV. All terminase subunits are essential for DNA packaging, but the roles of individual subunits are unclear. Until now, no viral protein has been identified that binds to the sites known to be required for initiation of the DNA-cleavage/packaging reaction. We have recently made a significant advance in purifying and characterizing pUL33. In preliminary data, we show that pUL33 binds to the pac2 sequence and speculate that this serves to initiate the DNA-cleavage/packaging process. Using methods developed in our laboratory, we propose to (i) characterize the DNA-binding activities of pUL33, (ii) map the functional domains of pUL33, and (iii) test whether the DNA-binding activity plays an important role in cleavage of DNA in infected cells. These studies will directly address an important issue in the field: how the terminase recognizes packaging signals in the viral genome to initiate the DNA-cleavage / packaging processes. In addition, studies on the mechanism of DNA-cleavage/ packaging may lead to development of novel anti-herpes virus strategies.